Kidney transplantation is the preferred treatment option for end stage kidney disease, but infectious and immunological complications limit the lifespan of kidney transplants. Urinary tract infections (UTI) occur at an alarmingly high rate after kidney transplantation and are associated with urosepsis, allograft loss, and mortality. The current gold standard for diagnosis of UTI is bacterial culture. Culture-based tests are, however, limited by their ability to detect only culturable bacteria, do not inform about co-infections and commensal microbiota, do not inform about bacterial growth dynamics, and do not inform about the host's response to infection. The goal of this proposal is to invent and apply genomic assays of urinary cell-free DNA that can simultaneously inform about the composition of the urinary microbiome, growth dynamics of pathogens associated with UTI, tissue injury in the graft, and the host's immune response to infection. A unique opportunity for this work is our large cohort of over 250 kidney transplant recipients in which we have serial biobanked urine specimens as well as routine conventional urine cultures in the first 3 months of transplantation. Using a newly developed single-stranded DNA library preparation technique, we will profile 290 urinary samples for cfDNA: i) serial samples from transplant recipients with UTI (prior to, during, and after UTI) ii) serial samples from recipients with recurrent UTIs; iii) samples from recipients with asymptomatic UTI and negative urinanalysis; and iv) samples from recipients who do not develop UTI in the first 3 months of transplantation. We will profile the dynamic urinary microbiome prior to, during, and after UTI in kidney transplantation. We will investigate whether cfDNA profiles are associated with UTI as well as whether they can anticipate UTI, UTI recurrence, and multi-drug resistant UTI. Based on the concept that bacterial genome replication rates and growth dynamics can be estimated from analyses of metagenomic sequence coverage, we will estimate growth dynamics over time and in response to antibiotic therapy in patients with UTI and recurrent UTI. We will monitor the host response to UTI via analyses of nucleosome footprints comprised within cfDNA and via donor specific cfDNA. Our pilot studies indicate that i) the structures of nucleosomal arrays in transcriptional regulatory elements are preserved and comprised within urinary cfDNA, and ii) that an analysis of nucleosomal occupancy in gene promoters can be used to quantify gene expression. We will investigate the differential host's response in subjects who have symptomatic UTI with positive urinalysis and asymptomatic UTI with negative urinalysis and the differential host's response in UTI patients after antibiotic treatment. We will assess tissue injury to the allograft in the scope of UTI, using donor specific cfDNA as a marker of allograft injury. Successful implementation of this proposal will provide a novel understanding of the pathogenesis of UTI as well as open new avenues for the utilization of cfDNA profiling as a sensitive and noninvasive transplant monitoring tool.